Recent advances in genetic engineering have given new impetus to crop improvement. Major breakthroughs have been achieved via development of genetic transformation techniques that facilitate introduction of heritable material such as deoxyribonucleic acid (DNA) into living organisms. Genetic transformation is a process which simply involves the uptake of foreign DNA by somatic cells of an organism. It is an unique mechanism by which foreign DNA of any origin (bacterial, animal, plant, etc.) is stably incorporated into the host genome. As a result, the introduced DNA becomes a part of the parent genome inflicting a permanent genetic change and can be inherited by the subsequent progeny, Chibbar and Kartha, 1994. Such human-engineered organisms harboring additional genetic information are called xe2x80x9ctransgenic organismsxe2x80x9d, whether they are plants or animals, Dekeyser et al., 1990.
Plant transformation techniques involving different systems of gene transfer have been a boon to plant breeders to introduce variability at the molecular level, Cocking and Davey, 1987, and thereby increase genetic diversity, Barton and Brill, 1983. It has helped in the creation of genotypes with novel traits. The recently released, extended shelf-life Flavr-Savr(trademark) tomatoes, Redenbaugh et al., 1992, transgenic Bt-cotton containing resistance to the cotton boll worm, Perlak et al., 1990, and many other such examples, have helped mankind save on costs due to fruit rot and insect control, to name a few. Therefore, transgenic plants could potentially provide an economic edge over conventionally bred crop species in terms of being environmentally friendly, in reducing the risks from using hazardous pesticides and herbicides, in addition to bolstering yields either directly or indirectly, Gasser and Fraley, 1992.
Although more is known about its genetics than many other crops, and despite extensive breeding efforts, maize (Zea mays L., Poaceae) as a crop, still requires continual genetic improvement. Examples include developing resistance (or tolerance) to diseases, pests, and herbicides, and improving the protein quality, all of which contribute to an increased net economic gain. Great emphasis has been placed on maize breeding for crop improvement due to its extreme value as a cereal crop worldwide, Chassan, 1992. In fact, maize ranks as the world""s third largest crop, trailing only behind wheat and rice, Langer and Hill, 1991. In the United States, it is the leading cereal (grain) crop and in 1995, 3,351,762 metric tonnes were produced on 26,314 hectares and was valued at greater than $23 billion, U.S. Department of Agriculture-National Agricultural Statistics Service, 1995-96. Due to the importance of maize in the U.S. and worldwide, many maize improvement programs are currently utilizing genetic engineering protocols to complement/enhance classical breeding efforts which are limited to working with genes (traits) already present in the maize germplasm.
The biolistic system has certain unique advantages over other gene transfer systems. It is very simple to operate and has universal applications because it can be used for gene transfer into cells of plants, animals, or microbes, Klein et al., 1992. Since its advent, it has been a very useful method commonly employed to transform the world""s cereal crops like rice, barley, sorghum, oats, wheat and maize, Christou et al. 1991; Klein et al., 1989; King et al., 1994; Ritala et al., 1994; Somers et al., 1992; Vasil et al., 1994. The biolistic process has since proven to be the best available system for transforming monocots, Batty and Evans, 1992.
Regeneration of maize involves the use of juvenile tissues by employing explants or tissues derived from seeds either pre- or post-germination. Successes have been limited to the use of embryogenic calli maintained in suspension cell culture (e.g. BMS and other elite inbred lines) initiated from immature embryos, and whole immature embryos as targets for developing biolistics-based transformation systems for maize, Gordon-Kamm et al., 1990; Fromm et al., 1990; Walters et al., 1992; Lowe et al., 1995. Ideal target tissues would be capable of accepting foreign DNA at high frequencies and regenerating plants (hopefully transgenic) efficiently.
Optimization must be conducted for each explant/tissue type and is usually based on the transient expression of the introduced genes. Transient expression refers to the expression of gene sequences that may or may not be integrated into the host genome and are usually conducted after a brief (42-72 h) post-bombardment period. Transient expression frequencies provide rapid and useful information as to whether foreign DNA was or was not introduced. In addition, correlations can be made to stable transformation frequencies. Researchers estimate the stable transformation frequency to be from less than 1% up to 5% of the transient expression frequencies, Finer and McMullen, 1990; Klein et al., 1988b. Thus, transient expression has proven to be a very useful indicator of DNA delivery and is routinely used in investigating the optimum conditions required to deliver DNA into different explant/tissue types via biolistics. For many crop species, optimization of bombardment (DNA delivery) is conducted by measuring the transient expression of the reporter gene, xcex2-glucuronidase (GUS), by a histochemical assay, 48-72 h post-bombardment, Jefferson et al., 1987.
It is evident that development of regeneration protocols for various target tissues and optimization of critical biolistic parameters utilizing these tissues assume great importance in developing a biolistics-based transformation system for maize.
Maize Tissue Culture
Maize has been regenerated in vitro by following different systems of regeneration. These include regeneration via somatic embryogenesis and organogenesis with both utilizing adventitious (including de novo) regeneration protocols, Vasil, 1986.
Immature Zygotic Embryos As Explants For Callus Cultures
The successful induction of somatic embryogenesis, maturation and germination of embryoids into plantlets are dependent on a number of variables. These variables include: genotype, age of the extracted immature zygotic embryos (days post-pollination; dpp), sucrose concentration in the media, the plant growth regulators (PGRs) utilized along with other media additives and associated culture conditions. Table 1 lists the genotypes successful in plant regeneration utilizing immature zygotic embryos as explants. Regeneration was a result of scutellar tissue proliferation which lead to the formation of embryogenic callus from which mature bipolar somatic embryos emerged and were subsequently regenerated into whole plantlets.
Seedling Nodal Tissues As Explants
Seedling nodal tissues from inbred B73 were excised from dark-grown, 3-4 day old germinated seedlings (obtained from mature seeds) and used to initiate organogenic callus cultures, Lowe et al., 1985. Nodal tissues were placed on a MS-based medium, Murashige and Skoog, 1962, containing 2% (w/v) sucrose, 0.5 mg/l 2,4-D, 3.0 mg/l picloram, 150 mg/l L-asparagine incubated under continuous light conditions at 28C. Shoot differentiation occurred on a MS-based medium containing 25 (w/v) sucrose and 10 mg/l kinetin under increased light intensity (10,000 lux). The shoots were transferred to a MS-based medium with 2% (w/v) sucrose and 0.1% (w/v) charcoal for rooting, Lowe et al., 1985.
Shoot Tip Apices As Explants
Raman et al., 1980, established shoot tip cultures as a method of maize propagation for eight genotypes (Table 3). Stem segment explants, which consisted of the shoot apical meristem plus a few axillary bud primordia, were excised from the scutellar region of 20 day old seedlings obtained from greenhouse grown maize. Those explants were used to enhance axillary bud proliferation (perhaps adventitious in nature) and developed maximum numbers of shoots when placed on a MS-based medium, Murashige and Skoog, 1962, containing 3% (w/v) sucrose, adenine sulfate dihydrate (120 mg/l), kinetine (3.0 mg/l) plus IAA or IBA (1.0 mg/l). It was determined that cytokinins alone did not stimulate axillary bud break/proliferation. NAA (5.0 mg/l) added to the basal medium (minus other PGRs) stimulated rooting of the excised shoots. In addition to genotypic differences in regeneration capacity, phenotypic abnormalities were noted in regenerated plantlets from all genotypes, Raman et al., 1990.
Somatic embryos and adventitious shoots were regenerated from shoot tips of maize seedlings and precociously germinated immature zygotic embryos from 18 genotypes, Zhong et al., 1992; Table 3. Mature seeds or immature zygotic embryos (10-15 dpp) were placed on numerous media in a step-wise manner, first on a MS-based, Murashige and Skoog, 1962, medium (A) containing sucrose (concentration not mentioned) and 500 mg/l of casein hydrolysate (CH) to achieve germination. After 7 days, the shoot tip explants were excised and placed on medium A plus 2.0 mg/l BAP for four weeks, then transferred to A containing 0.5 mg/l 2,4-D plus 2.0 mg/l BAP for four weeks to form adventitious shoots. For somatic embryo production, the cultures were then transferred to A containing 0.5 mg/l BAP. Further organogenic shoot development occurred on this same medium with rooting accomplished on A containing 0.87 mg/l IBA, Zhong et al., 1992. Explants from immature tassels and immature ears obtained from greenhouse-grown sweet corn hybrid Honey N Pearl plants could also initiate shoots on various media. Clumps of multiple shoots were regenerated from immature ears within four weeks when cultured on medium A supplemented with 2.0 mg/l BAP, and shoots formed on immature tassel explants when cultured on A containing 0.1 mg/l 2,4-D plus 1.0 mg/l BAP, Zhong et al., 1992.
Biolistics: History and Development
Introduction on micro-projectiles into tissues at high velocities were first used by plant vitologists to mechanically infect plants cells with naked non-infectious viral nucleic acids as an alternative to spraying the inoculum. These micro-projectiles wounded the plant cells and provided an entry for the viral nucleic acids which then could cause infection, MacKenzie et al., 1966. However, the credit for development of high-velocity micro-projectiles for genetic engineering purposes goes to Drs. J. C. Sanford, T. M. Klein, E. D. Wolf and N. Allen at Cornell University. They developed a number of devices that propelled DNA-coated tungsten micro-projectiles at high velocities into tissues which allowed DNA delivery into plant cells surpassing the cell wall, the primary barrier to DNA introduction. Their systems accelerated micro-projectiles via the following: gas discharge, transferred mechanical impulse, macro-projectile plus stopping plate, and centripetal acceleration, Sanford et al., 1987.
The macro-projectile-based system was most promising as it allowed the delivery of micro-projectiles into smaller cell sizes and did not have any detrimental impact on the target cells, Sanford, 1988. The device used a gunpowder charge to accelerate a macro-projectile which carried DNA coated micro-projectiles through a barrel towards a stopping plate. On providing the charge under partial vacuum, the micro-projectiles continued their acceleration into target plant tissues placed in their path. This gunpowder-based acceleration device was called the xe2x80x98particle gunxe2x80x99, Klein et al., 1987; Sanford et al., 1987. The inventors patented a modified version of the particle gun, called a xe2x80x98biolistic apparatusxe2x80x99, Sanford et al., 1989, and the bombardment procedure was referred to as the xe2x80x98biolistic processxe2x80x99, Sanford, 1988. Following their invention, several independent research groups constructed various DNA delivery systems such as the particle inflow gun (PIG; Vain et al., 1993), modified particle inflow gun, Gray et al., 1994, Accell(trademark) technology, McCabe and Christou, 1993, micro-targeting device, Sautter et al., 1991, and the simple particle bombardment device, Brown et al., 1994.
The era of biolistics as a tool for gene transfer was initiated when Klein et al., 1987, demonstrated the delivery of nucleic acids (both RNA and DNA) into living, intact Allium cepa L. (onion) cells. Tungsten micro-projectiles (4.0 xcexcm diameter) carrying TMV RNA were xe2x80x9cshotxe2x80x9d into onion epidermal cells at very high velocities, the first such report for plants, Klein et al., 1987. After bombardment, onion epidermal cells remained viable and those punctured by micro-projectiles coated with TMV RNA produced crystalline inclusions, an indication of the viral nucleic acid expression. Approximately 30-40% of the bombarded onion cells expressed the viral RNA. Onion epidermal tissues were also bombarded with tungsten micro-projectiles (4.0 xcexcm) coated with plasmid DNA encoding CAT, Klein et al., 1987. The CAT coding sequence contained the first intron of Adhl at its N-terminus flanked by CaMV 35S promoter and nos 3xe2x80x2 sequence. Three days post-bombardment, transient expression of CAT was demonstrated by using a radioactive precursor with products detected by thin layer chromatography, Klein, et al., 1987.
Sanford et al., 1987, proposed that the use of high velocity micro-projectiles would overcome problems associated with DNA delivery via protoplast-based methods like electroporation. Sanford, 1988, also opined that the biolistic process was an ideal gene delivery system because DNA delivery was simple and rapid. It allowed transformation of numerous competent cells and tissues of size as small as 5.0 xcexcm in diameter (plant/microbial cells, organelles) with the same basic protocol, irrespective of their shape and cell environment, Sanford et al., 1993. The DNA-coated micro-projectiles dispersed/scattered at random toward the targeted explant/tissues at very high pressures. A lack of uniformity in DNA delivery was inherent with the system. However, DNA delivery with very minimal damage to the target tissue was obtained. The biolistic process was then upgraded by making changes to the macro-projectile and stopping plate of the particle gun, Klein et al., 1987. These changes were incorporated into the gunpowder-based particle delivery system, PDS-1000 (licensed from DuPont with distribution via Bio-Rad), and the most recent helium-driven PDS-1000/He(copyright), Sanford et al., 1989. The gunpowder-based particle gun used nail gun cartridges as the power source. However, they were dangerous and left residues from gas and debris within the device. The upgraded helium-driven apparatus was safer and cleaner to use, Sanford et al., 1993.
PDS-1000/He(copyright) System For DNA Delivery
The PDS-1000/He(copyright) system is the only commercially available particle bombardment device (FIG. 1; Kikkert, 1993). The PDS-1000/He(copyright) contained a small, high pressure chamber with a gas acceleration tube that could be blocked by disks that ruptured at pre-set helium gas pressures. Once the pre-set pressure was reached, the helium would quickly enter the target chamber, which was maintained under vacuum. The target chamber contained a micro-projectile (micro-carrier) launch assembly which housed the macro-projectile and a stopping screen just beneath it. The helium pressure pushed the macro-projectile (macro-carrier) onto the stopping screen and the exerted force propelled the DNA-coated micro-projectiles off the macro-projectile and onto the target tissues.
During operation of the PDS-1000/He(copyright), in addition to alterations in helium pressure (450-2200 psi), other variables requiring optimization included the gap distance (0.32-1.0 cm; distance between rupture disk retaining cap and launch assembly), and target distance (3-12 cm; distance from stopping screen to target tissues). Higher helium pressures did not necessarily result in higher transformation rates, Sanford et al. 1993. Aside from altering helium pressures under vacuum, the macro-projectile and micro-projectile velocities could also be altered by changing the gap and target distances. Greater distances resulted in the micro-projectiles being scattered over a wider area of the target tissues.
Prior to loading samples onto the macro-projectiles and PDS-1000/He(copyright) operation, micro-projectiles of different type and size (gold: 0.6-1.6 xcexcm; tungsten: 0.4-1.7 xcexcm) could be mixed with the chosen DNA (xe2x89xa7one plasmid type). The amounts of micro-projectiles and DNA used per shot and the number of shots per tissue could also be varied. Although the system was flexible in that many parameters could be varied, each needed to be optimized (in combination with other parameters) for each explant/tissue type to obtain the greatest transient and stable transformation frequencies, Klein et al., 1988c.
Genes For Introduction Into Monocots
The vectors or gene constructs used for biolistic experiments should encode appropriate reporter and selectable marker genes capable of expression either constitutively or in specific cell types. Gene constructs used for monocots usually contained monocot promoters and a monocot intron fused to N-terminal region of the coding sequences. Monocot introns have been demonstrated to significantly enhance gene expression in monocots, Callas et al., 1987; Vasil et al., 1989; McElroy et al. 1990; Last et al., 1991; Mass et al., 1991 and Donath et al., 1995. Klein et al., 1988a, detected high CAT activity in BMS cells bombarded with pCaMVI1CN harbored the first intron of the Adhl fused to N-terminal region of CAT controlled by CaMV 35S promoter and nos 3xe2x80x2 sequences. Decreased levels of CAT expression were noted when BMS cells were bombarded with pCaMVCN, an identical CAT-based construct except it lacked Adhl intron, Klein et al., 1988a. A construct with the first intron of Adhl fused to the N-terminal region of GUS controlled by the Adhl promoter with nos 3xe2x80x2 sequences was demonstrated to enhance GUS expression in suspension cultures of A188 X B73 and B73 X A188, Fromm et al., 1990.
Higher transient expression rates were noted in suspension cells of Panicum maximum (guineagrass) cv. Pm86, maize (cv. Zm85), Pennisetum purpureum (napiergrass) cv. Pp90, Pennisetum glaucum (pearl millet) cv. Pg86, Saccharum officinarum (sugarcane) cv. Sc84, wheat cvs. Ta87, TA89, and TA90, and immature embryos of pearl millet following bombardment via the gunpowder-based PDS-1000 when plasmid pAHC25 was utilized versus pBARGUS, Taylor et al., 1993.
Plasmid pAHC25 contained GUS and bar coding sequences driven by maize ubiquitin (Ubil) promoters and nos 3xe2x80x2 with the first intron of Ubil fused to N-terminal regions of GUS and bar. Plasmid pBARGUS contained GUS and bar and coding sequences with Adhl intron 1 fused to each N-terminal region. Flanking regions included Adhl promoter (GUS), CaMV 35S (bar) and nos 3xe2x80x2. Similar results were obtained in comparisons utilizing three week old embryogenic callus cultures of wheat cvs. Bob White, Pavon and RH770019 following particle bombardment DuPont PDS-1000 or PDS-1000/He(copyright), Vasil et al., 1993.
Superiority of GUS expression in transgenic tissues containing pAHC25 was attributed to the ubiquitin promoter which was constitutively expressed irrespective of the tissue type, while GUS in pBARGUS controlled by the Adhl promoter was demonstrated to be developmentally regulated, organ specific, and up-regulated under anaerobic conditions, Taylor et al., 1993. Ubiquitin-based plasmids were therefore considered superior to the Adhl-based plasmids, Taylor et al., 1993; Vasil et al., 1993. However, studies using Sorghum vulgare L., cv. Grazer suspension cultures bombarded with plasmids containing NPTII, hph, and GUS genes driven by the Adhl promoter determined that the monocot promoter yielded higher transient GUS expression levels compared to those genes driven by CaMV 35S promoter, Mendel et al., 1989.
Suspension cultures derived from immature zygotic embryos of barley, winter type cv. Borwina bombarded with NPTII- and GUS-containing plasmids revealed that plasmid size was also a critical factor to be considered, Hagio et al., 1991. Bombardment with a smaller plasmid (5.1 kilo bases; kb) yielded more stable transformants versus larger ones (12.8 kb and 14.2 kb). However, the amounts of plasmid DNA delivered per shot were not equal with respect to all three plasmids, Hagio et al. 1991.
Transient Expression Quantified By GUS Assays
As mentioned previously, the target explants/tissues were usually bombarded with a reporter gene to measure its expression visually, an easy technique to confirm delivery of the foreign DNA. Initial biolistic experiments conducted by researchers utilized the xcex2-glucuronidase (GUS) gene as a xe2x80x98reporterxe2x80x99 of successful DNA delivery and its gene product was assayed histochemically as xe2x80x98bluexe2x80x99 cells, Wang et al., 1988; Klein et al., 1988c. This proved to be a convenient measure to optimize the gene transfer efficiency in rice, wheat and Glycine max (soybean), Wang et al., 1988. The GUS xe2x80x98blue cellxe2x80x99 assay was also successfully used to detect foreign DNA in bombarded tobacco suspension cultures from line XD, Klein et al., 1988b. In deciphering the factors or parameters that influenced gene delivery into BMS suspension cultures via transient expression, Klein and coworkers, used GUS to monitor DNA introduction via transient expression assays, Klein 1988c. The plasmid pAI1-GusN contained the Adhl promoter, the first intron of Adhl fused to the 5xe2x80x2 end of the GUS coding region and nos 3xe2x80x2. From 1988, the GUS assay formed an integral part of many biolistic experiments to optimize the biolistic parameters based on transient expression and to achieve high transformation efficiencies, Klein et al., 1988c.
The expression of GUS was detected histochemically when the bombarded cells were incubated in the presence of a synthetic substrate, 5-bromo-4-chloro-3-indoyl-xcex2-D-glucuronic acid (X-glu). The cells turned blue only when the substrate was cleaved by xcex2-glucuronidase enzymatically, thus confirming the introduction and expression of the GUS gene. The blue color was due to the formation of a precipitate which could be visualized, Jefferson et al., 1987. Some researchers have also used luciferase or anthocyanin genes as reporter genes, King and Kasha, 1994; Goff et al., 1990; Fromm et al., 1990.
In U.S. Pat. No. 5,320,961, Zhong, et al., a method for asexually propagating maize to produce a fertile corn plant is described in which shoot tip apices isolated from either caryopses or kernels are employed. The explant relied on by Zhong consists of five mm sections of the localized enlargement of the seedling at the junction of the mesocotyl and the leave sheath. The explant described essentially contains the shoot tip, three to five leave primordia, and a portion of young leaf and stem immediately below the leaf primordia. This indicates that the explant employed by Zhong is green (because the seedling is one week old, the localized enlargement would be dark green which gives an indication of its advanced developmental stage; this is a characteristic feature of reduced cell division combined with enhanced cell elongation).
Zhong does not describe the transformation of maize tissue by the introduction of exogenous DNA. To ensure active uptake of exogenous DNA, it is important that the target tissue be in an early developmental stage, such that the tissue is comprised of cells that are in a state of cell division and not cell elongation.
Zhong et al. employ mature seeds or precociously germinated immature zygotic embryos (10-15 days post-pollination) placed in numerous media in a step-wise manner, first on a MS-based medium containing sucrose and casein hydrolysate to achieve germination, followed by the medium further containing 2.0 mg/l 6-benzylaminopurine (BAP). The Zhong process is plant growth regulator (PGR) dependent, and takes about 17-19 weeks to obtain plantlets.
Accordingly, it remains an object of those of skill in the art to provide a method to asexually regenerate maize, and to couple such a regeneration process with a transformation process to introduce exogenous, desirable DNA, which is preferably genotype-independent, and generally applicable to a wide variety of commercially important crops. Accordingly, the regeneration protocol must rely on tissue actively in the state of cell division.
Regeneration and Transformation of Corn
The corn transformation procedure of this invention integrates a corn multiple shoot induction protocol with nodal sections as explants. Those explants are also used as targets in a biolistics-based transformation system.
Surface Sterilization and Germination of Maize Seeds
Corn seeds are surface sterilized in a solution containing 20% (v/v) commercial bleach and 0.5% SDS for 15 min. under continuous shaking, then serially rinsed in sterile double-distilled water (sddw) four to five times. Liquid MS-based germination medium (CSG) containing MS salts, sucrose (30 g/l), DM-vitamins (1.0 mg/l thiamine-HCl, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine-HCl and 100 mg/l myo-inositol) and BAP (2.0 mg/l) at pH 5.8 is dispensed per Magenta(trademark) box (45 ml) containing eight layers of cheese cloth, then autoclaved. Seeds are placed in CSG (25 seeds of any genotype per box) and cultured for three days (16 h or continuous light; 25C) for germination. Nodal section explants (one per seedling) are excised from seedlings approximately 3-10 days old, preferably from 3-7 days old. The nodal section appears as a clear demarcation on the germinating seedling and represents the seventh node. Cuts are made just above and below the node resulting in a 1.2-1.5 mm length cross-section. Thus, the explant employed is in an early developmental stage, indicating that the tissue (at least 50%) is comprised of cells that are surely in a state of cell division and cell elongation (Kiesselbach, Research Bulletin 161, University of Nebraska (1980)).
Multiple Shoot Induction
Nodal section explants are placed on corn shoot induction medium [CSI; MS salts, sucrose and DM-vitamins same as above, BAP (2.0 mg/l; filter-sterile, incorporated post-autoclaving), CPA (0.25 mg/l; filter-sterile, incorporated post-autoclaving), glycine (10 mg/l) and asparagine (150 mg/l) and phytagar (8 g/l) at pH 5.8], acropetal end up, and placed under the culture conditions previously mentioned. Tissues are subcultured every two weeks onto fresh CSI medium for multiple shoot formation. Adventitious shoots are separated from the shoot clumps after eight weeks and elongated on a semi-solid MS-based medium containing sucrose, DM-vitamins, glycine (10 mg/l) and asparagine (150 mg/l) at pH 5.8 for three weeks (FIG. 1E) or shoot elongation mediums modified with various plant growth regulators. The plantlets are rooted on the same medium but which also contains indole 3-bytyric acid (IBA) (0.5 mg/l). Rooted plantlets can be grown in PGR-free liquid MS in test tubes (150xc3x9725 mm) containing cheesecloth as the anchor material to achieve faster growth. Regenerated plantlets are transplanted to potting media, acclimatized then grown to maturity in the greenhouse.
Preparation of Tungsten Microprojectiles
Approximately 60 mg of tungsten microprojectiles [M-10 (0.7 xcexcm) or M-25 (1.7 xcexcm)] is weighed and placed in a 1.5 ml microcentrifuge tube with 1.0 ml 100% ethanol, then vortexed vigorously for 3 min. The mixture is microcentrifuged for one min., the supernatant discarded and the ethanol wash procedure is repeated twice. The microprojectiles are then resuspended in 1 ml 100% ethanol and placed at room temperature overnight. The following day, the mixture is microcentrifuged for 1 min. The supernatant is removed and the microprojectiles are washed in 1.0 ml sddw and microcentrifuged for 1 min. The supernatant is discarded and the microprojectiles are then resuspended in 1.0 ml sterile 50% (v/v) glycerol yielding a 60 mg/ml stock that can be stored frozen (xe2x88x9220C) before use.
Sterilization of Macrocarriers and Rupture Disks
Macrocarriers and rupture disks (450, 650, 900, 1100 or 1350 psi) are sterilized for 15 min in 100% ethanol and dried in the laminar air flow hood and the gene gun parts are sterilized overnight in 70% ethanol. The gene gun is disinfected by a thorough spray of reagent alcohol.
DNA Precipitation Onto Tungsten Microprojectiles
On ice, approximately 5 xcexcl (1.0 xcexcg/xcexcl) nuclear transformation vector pAHC25 DNA is added to 60 xcexcl tungsten microprojectiles in a Treff tube. The following are then added: 10 xcexcl isopropanol (2xc3x97 vol of DNA), 50 xcexcl 2.5M CaCl2 and 20 xcexcl 0.1M spermidine and vortexed for three min. The mix is incubated on ice for three min. then the vortex and incubation steps are repeated. The mixture is then microcentrifuged and the supernatant discarded. The microprojectiles are washed once in 250 xcexcl 70% ethanol and microcentrifuged for one min. The supernatant is discarded and the microprojectiles are resuspended in 60 xcexcl 100% ethanol and vortexed briefly. The DNA loaded microprojectiles can also be maintained frozen pending use.
Approximately 12 xcexcl of the DNA-coated microprojectiles are loaded onto each macrocarrier which delivers 1.0 xcexcg DNA and 720 xcexcg of tungsten per shot. The above mixture is good for approximately five shots.
Bombardment of Nodal Sections
On the day of bombardment, nodal sections are excised and placed, acropetal end up, in the central 2.5 cm area of a petri dish (40 per dish) containing CSI medium devoid of amino acids.
Bombardment Parameterss
The following parameters are used to bombard corn nodal section explants: 650, 900, 1100, or 1350 psi helium pressure; gap distance (1.0 cm); and target distance (7.5 cm), with two bombardments per plate using the PDS-1000/He device (Bio-Rad). M10 (0.7 xcexcm) or M25 (1.7 xcexcm) tungsten microprojectiles were used in bombardments.
Selection of Transgenic Tissues Post-Bombardment
The nodal sections are separated and spread out on CSI medium maintaining polarity and placed in the dark for two days post-bombardment. Histochemical staining for xcex2-glucuronidase expression was conducted three days post-bombardment (FIG. 1C). Explants are then placed on CSI medium devoid of amino acids but contains the selective agent, phosphinothricin at 2.0 mg/l. The regeneration protocol described above is followed to select for green shoots and rooted in the presence of phosphinothricin. Transgenics are confirmed via PCR analysis with appropriate primers specific for the bar and xcex2-glucuronidase sequences.
This invention resides in the discovery that novel explant sources, nodal sections excised from germinated embryos and germinated mature seeds, principally characterized by being comprised of cells in a state of cell division, approximately 3-10 days old, preferably from 3-7 days old, can be used to promote regeneration of fertile plants. Simultaneously, the invention herein resides in the development of a process for biolistic transformation which is independent of explant source and genotype. To demonstrate the universal suitability of the biolistic transformation protocol that is one aspect of the invention herein, not only nodal explants of the invention herein, but two other target tissues, including immature zygotic embryos (approximately 13-15 days post pollination or DPP) and mature zygotic embryos were selected as target tissues for biolistic exogenous DNA bombardment. The regeneration of zygotic embryos, and their isolation, per se, as target tissues for transformation, does not constitute an aspect of this invention, and development of protocols to optimize that regeneration are not discussed below. Prior art protocols for these two previously explored target tissues have been developed, and may be equally used. Accordingly, the invention lies in the development of a protocol for regeneration of maize using young nodal sections as explants, coupled with universally applicable biolistic DNA bombardment transformation protocol.
In the experiments described below, a wide variety of parameters are explored, particularly with respect to transformation processes. Many of these parameters are numerically expressed or quantified. Given the need to demonstrate genotype and tissue independence, only so many numerical values may be explored. Nonetheless, the numerical it values represent ranges judicially selected about the selected parameters, which ranges appear in the claims appended to this application. The examples provided are not intended to be limiting, and the ranges recited are based on experimental observation, coupled with prior experimentation in related protocols and plants.
In the discussion set forth below, regeneration from nodal sections is first discussed. Having established that nodal explants from germinated embryos and germinated mature seeds can be effectively regenerated into fertile plants, DNA transformation techniques are then explored. In practice, the nodal explant targets are selected, transformed, and regenerated, to give new and improved maize genotype/fenotype crops.
Materials and Methods
All experiments were conducted in a sequential order, in that the cell/tissue culture and regeneration protocols were first developed followed by optimization of gene delivery using the PDS-1000/He(copyright). Tissue culture protocols were established utilizing nodal sections each composed of explants from two different developmental/physiological stages. They included nodal sections excised from aseptically germinated seedlings excised from mature seeds or from germinated immature zygotic embryos.
Maize seeds of all genotypes were provided by Dr. W. P. Williams (Supervisory Research Geneticist, USDA-ARS, Starkville, Miss.). Seeds were field-planted and also grown in greenhouses during the off-season to obtain explants (immature zygotic embryos) for the experiments. Field/greenhouse space and maize production assistance were provided by Dr. W. P. Williams (USDA-ARS) and Dr. Brian S. Baldwin (Assistant Professor, Department of Plant and Soil Sciences, Mississippi State University). Twenty-one different genotypes of maize including 16 grain hybrids, one sweet corn hybrid, and four inbreds were used for confirmation of all developed protocols. Table 5 provides the list of genotypes evaluated in optimization of regeneration and used for optimization of gene delivery via PDS-1000/He(copyright).
Immature Ear Production in the Greenhouse
Greenhouse plantings were undertaken at two different locations, at the USDA greenhouse (located near the Boll Weevil Research Unit, Starkville, Miss.) and at the Plant Science Research Center (PSRC), Mississippi Agricultural and Forestry Experimental Station (MAFES) greenhouse (North farm, Mississippi State, MS).
In the USDA greenhouse, the initial planting occurred in early October, 1995. Four maize seeds were planted per 9.45 liter plastic pot containing a potting mix with equal parts of sand and Bacto potting media, then thinned to retain one plant per pot. Two pots per genotype were maintained for all 21 genotypes. Subsequent plants (two plants per genotype) were staggered for adequate pollen supply. The greenhouse temperature was maintained between 23-31C. Sodium vapor lights in the greenhouse provided the light intensity required for growth and development of maize. A 12 h photoperiod was maintained until the plants tasseled and produced ears. The plants were initially watered twice daily then once per day, and fertilized two weeks post-emergence and every four weeks after establishment with 14-14-14 osmocote time-released fertilizer (2.0 g per application). Insect-control sprays were undertaken on an as-needed basis using Dipel for lepidopteran pests and Knox Out (Diazinon aerosol) for aphid control.
In the MAFES-PSRC greenhouse, the plants for all 21 genotypes were initiated in late February 1996. The maize seeds were planted in 7.56 liter plastic pots containing a mixture of sand, potting soil and ERTH Food (ERTH Group Inc., 1:1:1). The greenhouse temperature was maintained at 28xc2x13C. Four seeds per pot were initially planted and thinned to one plant per pot, post-germination. Two plants were maintained per genotype for all 21 genotypes. Subsequent plants (two plants per genotype) were staggered. No external lighting was provided and watering was once per day. The plants were first fertilized after emergence at the 3-4 leaf stage and every 15 days with 200 mg/l of ammonium nitrate until maturity and harvest.
Maize seeds in the USDA field trials (MAFES-PSRC, North farm) were planted in April, 1996 in rows of 406 cm. Each hybrid/inbred was planted as a separate designated row. Approximately 15 plants were maintained per row with 96.5 cm spacing between each row. The field was supplied with a basal nitrogen dose of 110 kg/ha. Herbicides, Dual (metolachlor) and atrazine at 2.5 kg/ha each were also incorporated to achieve weed control. The field was irrigated in furrows at weekly intervals only if rains were not received. Another top dress of nitrogen (168 kg/ha) was supplied 40 days post-planting. Linuron, a post emergence-herbicide was applied 65 days after planting (1.7 kg/ha).
Hand-Pollination and Harvest
The plants were monitored closely for initiation of tassels and silks. The ear shoots were covered with white paper bags prior to silk emergence. When the tassels dehisced pollen and silks were receptive to pollen, tassels were covered with brown paper bags and plants were self-pollinated by following standard hand pollination procedures. Hand pollination was done in the morning hours between 8:30 to 11:30 a.m. to ensure good kernel set. The tassels of a genotype were shaken completely within the brown paper bag to facilitate the release of pollen. The pollen in the bag was dropped onto the stigma (on one or two silks) of the same genotype (white bag off), then covered with the brown bag and stapled at the bottom carefully to avoid any foreign pollen contamination. Ears were allowed to develop within the brown paper bags following pollination (date noted on bag). The ears were allowed to develop only for a restricted period of time following self-pollination. Ears were harvested 12, 14 and 18 days post-pollination (dpp). Immature cobs were either used to extract embryos that were placed in culture immediately or stored in a refrigerator at 4C for up to three weeks for further use in experimentation.
Media Composition and Culture Conditions
The media composition and culture conditions were varied depending on the explant type and the regeneration type desired. The media used in the experiments contained either full strength MS, Murashige and Skoog, 1962, or N6, Chu et al., 1975, basal salts (4.3 g/l or 4.0 g/l respectively; Sigma), DM-vitamins (1.0 mg/l thiamine-HCl, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine-HCl, and 100 mg/l myo-inositol) with varying sucrose concentrations but constant phytagar concentration [0.8% (w/v); GibcoBRL]. It should be noted that vitamins other than DM-vitamins can be used as well. Additionally, it should be noted that the basal salts are not limited to any particular kind or type. Some media also contained filter-sterile amino acids L-proline (Sigma), glycine (Sigma) and L-asparagine (Sigma) which were incorporated into the medium after autoclaving. Stock solutions of L-proline (700 mg/ml), glycine (10 mg/ml) and L-asparagine (150 mg/ml) were prepared accordingly and filter-sterilized with a 0.2 xcexcm filter (Sterile Acrodisc(copyright), Gelman Sciences) and stored at 4C. All media were adjusted to the desired pH prior to autoclaving and the pH of the media varied with regeneration type. All media were autoclaved for 35 min. at 121C and 100 kPa pressure. Filter-sterile plant growth regulators (PGR) were incorporated into the media after autoclaving. Each petri plate (100xc3x9720 mm, Nunc) contained approximately 20 ml of medium. The stock solutions of PGRs [2,4-D, BAP, BA, zeatin, kinetin, 4-chlorophenoxyacetic acid (CPA), and GA3] were prepared at 1.0 mg/ml, filter-sterilized using a 0.2 xcexcm filter and stored at 4C or xe2x88x9220C. Depending on the type of regeneration, the cultures were either incubated in the dark at room temperature or in a Percival growth chamber (model 135LLVL, Iowa) under cool white fluorescent lights with a light intensity of 52 xcexcEmxe2x88x922sxe2x88x921, maintained at a 16 h photoperiod with 25/23C (internal) day/night temperatures.